The combination of immunological and microfluorimetric techniques has permitted the identification and analysis of the Ca2+ influx responses in single rat midbrain cholinergic terminals. These terminals represent 22% of the total synaptosomal population and about 63% of them responded to nucleotides by a Ca2+ influx. The nucleotide response distribution in cholinergic synaptic terminals is as follows; 22.4% to diadenosine pentaphosphate (Ap5A), 24.7% to adenosine 5'-triphosphate (ATP) and 16.3% to both agonists. The ATP and Ap5A are able to induce acetylcholine release in a dose- and calcium-dependent way, being the EC50 values 0.22+/-0.1 microM and 1.5+/-0.1 microM respectively. Specific inhibitors can block this secretory effect. The studies of Ca2+ influx responses in isolated single synaptic terminals have also permitted to demonstrate the wide co-expression of functional nicotinic and nucleotidic receptors. The percentage values of the terminals responding to both ATP/nicotine and Ap5A/nicotine were 18.4% and 19.1%, respectively, considering the total population. Immunological studies also confirmed the presence of P2X3 subunits and alpha4 and alpha7 nicotinic receptor subunits in about 36%, 30% and 20%, respectively, of the cholinergic terminals.